Intermediate filaments (IF; also called glial filaments in astrocytes) are a major cytoskeletal component of astrocytoma cells. Several transfection experiments with sense and anti-sense cDNAs have shown that IF proteins influence the malignant behavior of astrocytoma cells. However, the mechanisms responsible for this effect are unknown. Our preliminary results suggest that one of these mechanisms may involve the IF protein synemin. We found that synemin is frequently expressed by astrocytoma cells in brain tumors, but not by astrocytes in normal, adult brain. Intriguingly, in astrocytoma cells, synemin, unlike other IF proteins, can be present within structures related to cell motility, such as the lamellipodium and focal contacts. Synemin is also unique among IF proteins in that it has been shown to bind to actin-associated proteins such as alpha-actinin. Interestingly, we found that the association of synemin with the lamellopodium is regulated by TGF-alpha, a growth factor present in the microenvironment of astrocytic tumors. Indeed, in U373 glioblastoma cells maintained in serum-free medium, TGF-alpha induces synemin relocation from the IF network to the lamellipodium, an event which correlates with increased cell motility. Altogether these findings lead us to hypothesize that synemin, due to its unique binding properties and dynamic behavior can influence the organization and mechanical properties of cytoskeletal networks in astrocytoma cells. This would affect cellular features dependent on these properties, such as motility and cell shape. Specific aspects of this hypothesis will be examined in Aims 1 and 2. In Aim 1, we will "Investigate the Mechanisms by which TGF-alpha Regulates the Dynamic Properties of Synemin in Astrocytoma cells" by examining the hypothesis that phosphorylation plays a role in directing synemin to the lamellipodium. In Aim 2, we will investigate "How Synemin/alpha-Actinin Interactions Affect the Organization and Mechanical Properties of the Cytoskeleton" by determining whether synemin crossbridges IF and/or actin filaments and how this crossbridging may affect the visco-elasticity of these biopolymers. Finally, one of the obstacles in understanding the function of IF in astrocytoma cells is our limited knowledge of the binding partners of GFAP, the astrocyte-specific IF protein. We thus propose in Aim 3 to "Identify Proteins Interacting with GFAP in Astrocytoma Cells by Using the Yeast Two-Hybrid System".